RESEARCH ARTICLE

A conserved axon type hierarchy governing peripheral nerve

assembly
Liang Wang
1
, Alessandro Mongera
2
, Dario Bonanomi
3
, Lukas Cyganek
1
, Samuel L. Pfaff
3
,
Christiane Nu sslein-Volhard
2
and Till Marquardt
1,
*
ABSTRACT
In gnathostome vertebrates, including fish, birds and mammals,
peripheral nerves link nervous system, body and immediate
environment by integrating efferent pathways controlling movement
apparatus or organ function and afferent pathways underlying
somatosensation. Several lines of evidence suggest that peripheral
nerve assembly involves instructive interactions between efferent and
afferent axon types, but conflicting findings challenge this view. Using
genetic modeling in zebrafish, chick and mouse we uncover here a
conserved hierarchy of axon type-dependent extension and selective
fasciculation events that govern peripheral nerve assembly, which
recapitulates the successive phylogenetic emergence of peripheral
axon types and circuits in the vertebrate lineage.
KEY WORDS: Axon guidance, Axon-axon interactions, Motor
neurons, Peripheral nerve, Sensory neurons, Sympathetic neurons,
Mouse, Chick, Zebrafish
INTRODUCTION
Nervous system evolution seems to have frequently proceeded via
the use of pre-existing axon pathways, rather than through de novo
formation of nerve tracts, to accommodate novel features within
extant circuits (Katz, 1983; Katz et al., 1983). The segmental
organization of the vertebrate body axis, for example, places
constraints on peripheral axon growth that force primary
somatosensory axons (SAs) to extend through peripheral nerve
tracts that are also occupied by more ancestral motor efferent axons
(MEs) (Bonanomi and Pfaff, 2010; Butler and Hodos, 2005). Such
co-confinement to narrow substrate corridors may effectively foster
interactions between axons that extend from phylogenetically newer
or older neuron types, and accelerate their incorporation into
common functional assemblies.
Indeed, peripheral nerve assembly has long been thought to
involve prerequisite association of SAs with earlier extending MEs
en route to peripheral targets (Hamburger, 1929; Taylor, 1944;
Honig et al., 1986; Swanson and Lewis, 1986; Honig et al., 1998).
For example, surgical or laser-mediated removal of ventral neural
tube segments, including motor neurons, in amphibian and avian
embryos was observed to frequently result in the development of
aneural limb muscle (Hamburger, 1929; Honig et al., 1986;
Swanson and Lewis, 1986; Taylor, 1944). Our own recent data
provided a mechanistic basis for some of these ideas by showing
that, in mouse, SAs are guided to targets in the dorsal trunk by
molecular labels on earlier extending epaxial MEs (Wang et al.,
2011). Genetic manipulations that completely blocked ME
extension resulted in randomized extension of SAs along dorsal
or ventral trajectories, whereas ME-restricted elimination of the
EphA receptor tyrosine kinases EphA3 and EphA4 triggered
selective dorsal-to-ventral misrouting of SAs (Wang et al., 2011;
Wang and Marquardt, 2013).
Other recent data on mouse limb innervation, however, support a
minimal cooperative model according to which mutual interactions
between MEs and SAs have only a limited influence on the
establishment of peripheral nerve trajectories (Huettl et al., 2011).
For example, upon partial genetically induced elimination of MEs in
mouse embryos, only mild SA extension defects were observed,
whereas elimination of DRG neurons or DRG neuron-derived
neuropilin receptor expression led to pronounced defasciculation,
but not to mistargeting, of MEs (Huettl et al., 2011). Studies based on
surgical manipulations in frog and chick embryos arrived at yet
different conclusions, proposing that establishment of limb SA and
ME trajectories can be entirely dissociated fromeach other (Wang and
Scott, 1999; Wenner and Frank, 1995). Consolidating these conflicting
lines of evidence remains difficult because differences in animal
models, methodologies and positional identities of the peripheral
nerve segments studied currently preclude their direct comparison.
RESULTS
Conserved reliance of sensory axon extension on pioneer
motor axons
To explore the interactions between primary somatosensory afferent
axons (SAs) and motor efferent axons (MEs) we first systematically
investigated the relationships between molecularly identified
peripheral axon types in three different vertebrate species: zebrafish
(D. rerio: Fig. 1A), chick (G. gallus domesticus: Fig. 1J) and mouse
(M. musculus: Fig. 1S). In anamniotes, including zebrafish, the first
emerging sensory-motor circuits are dedicated to simple larval escape
reflexes that are mediated by an early central nervous system (CNS)
neuron population, Rohon Beard cells (RBs), which feed primary
sensory inputs from dermis to motor neurons controlling trunk
musculature (Spitzer, 1984). RBs are eventually replaced by neural
crest-derived dorsal root ganglion (DRG) neurons that become
incorporated into circuits facilitating a wider spectrum of motor
outputs (Butler and Hodos, 2005; Dasen, 2009; Holland, 2009). In
zebrafish, these circuit rearrangements are reflected by a 20 h delay
between initiation of primary ME extension, visualized by a motor
neuron-specific regulatory module of the NBT (Xenopus -tubulin)
gene to drive red fluorescent protein expression (dsRed) (Peri and Received 19 November 2013; Accepted 2 March 2014
1
Developmental Neurobiology Laboratory, European Neuroscience Institute
(ENI-G), Grisebachstrae 5, Go ttingen 37077, Germany.
2
Department of Genetics,
Max-Planck Institute for Developmental Biology, Spemannstrasse 35, Tu bingen
72076, Germany.
3
Gene Expression Laboratories, Howard Hughes Medical
Institute, The Salk Institute of Biological Studies, 10010 N. Torrey Pines Road,
La Jolla, CA 92037, USA.
*Author for correspondence (t.marquardt@eni-g.de)
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any mediumprovided that the original work is properly attributed.
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Nusslein-Volhard, 2008), and the emergence of SAs from DRGs,
visualized by exploiting the DRG neuron-restricted activity of the
neurogelin 1 promoter to drive green fluorescent protein (GFP)
expression (Blader et al., 2003) (Fig. 1B-E; supplementary material
Fig. S1A-E). SAs thus invariably extended along preformed
ME trajectories.
To visualize SAs and MEs in chick, we used previously identified
enhancer modules of the Isl1 and Hb9 genes (Lee et al., 2004) to
confine green and red fluorescent protein expression to DRG and
motor neurons, respectively (supplementary material Fig. S2A-F). In
mouse, DRG and motor neurons axons were respectively visualized
by the previously established DRG and motor neuron-specific
transgenes Brn3a
tau:lacZ
and Hb9::eGFP (Lee et al., 2004; Trieu
et al., 2003). In amniotes, including chick and mouse, formation of
larval Rohon Beard circuits is skipped, and primary sensory-motor
circuits directly assemble from motor neurons and DRG neurons
(Dasen, 2009; Holland, 2009). Despite this altered configuration,
the principal chronological sequence of peripheral axon extension
observed in zebrafish appears to be preserved in chick and
mouse: SAs invariably extended along trajectories pioneered
by MEs, as reported previously (Fig. 1K-N,T-W; supplementary
material Fig. S2A-F) (Honig et al., 1986; Wang et al., 2011).
We next tested whether this rigid axon type-dependent extension
order would reflect a conserved primacy of the first-extending MEs
during peripheral nerve assembly. To address this, we studied SA
extension upon preventing ME extension. In zebrafish, this was
achieved by injecting morpholino oligonucleotides targeting the
Isl1 gene that were previously found to selectively interfere with
motor neurogenesis (Hutchinson and Eisen, 2006). The resulting
absence of MEs led to severely reduced initial extension rates
of SAs (Fig. 1F-I) and frequently triggered highly erratic patterns of
SA extension (supplementary material Fig. S1L-Q). In chick, the
prevention of ME extension, by introducing Hb9-driven cell-
autonomous diphtheria toxin (DTA) into the neural tube prior to
motor neurogenesis, resulted in a randomized loss of dorsal or
ventral trunk SA pathways within the confines of normal peripheral
nerve trajectories (supplementary material Fig. S2G-J), resembling
those previously reported by us upon DTA-mediated ablation of
motor neuron progenitors in mouse (Wang et al., 2011).
We next asked whether the apparently conserved role of MEs in
establishing trunk SAtrajectories would similarly govern assembly of
peripheral nerve pathways that co-evolved with the tetrapod limb
(Butler and Hodos, 2005; Luria et al., 2008; Ma et al., 2010). Similar
to zebrafish, genetic ablation of motor neurons or their progenitors
prior to ME extension in chick and mouse (supplementary material
Figs S2G,H and S3A-J) resulted in markedly reduced SA extension
rates (Fig. 1O-R,X-AA). In contrast to the effects of ME removal on
chick and mouse trunk innervation, where SAs projected in a
randomized manner within largely normal peripheral pathways (see
supplementary material Fig. S2I,J) (Wang et al., 2011), lumbar SAs
exhibited highly aberrant projections that frequently deviated from
the trajectories normally chosen by peripheral axons (Fig. 1Q,Y,Z).
These defects culminated in the failure or severe delay of most SAs to
extend beyond the limb plexus (Fig. 1Q,R,Z,AA; supplementary
material Fig. S3K,L), possibly owing to the markedly delayed SA
extension. Under conditions of delayed or partial prevention of ME
Fig. 1. Conserved reliance of sensory afferent axon (SA) extension on pioneer motor efferent axons (MEs). (A) Peripheral axons visualized in 72 hpf
zebrafish larva. (B-E) Time-lapse sequence of SA, labeled using -8.4neurog1::GFP transgene (yellow), extending peripherally along preformed CaP ME,
labeled by NBT::dsRed transgene (magenta). Arrowheads indicate SA growth cone. (F-H) Delayed and aberrant SA extension in the absence of MEs upon
islet1E2/E3 morpholino injection. (I) Longitudinal analysis of SA and ME extension and impacts of ME removal (n7 segments, 3 embryos per stage and
condition). (J) Peripheral axons visualized in E4 chick embryo. (K-N) SAs, labeled using the Isl1
DRG
::mGFP transgene (yellow), extending along preformed
MEs, labeled by co-transfected Hb9
MN
::mCherry. Arrowheads indicate distalmost SA growth cone. Asterisks indicate additional Isl1
DRG
::mGFP activity in
non-DRG neural crest cells. Dotted line indicates trunk/limb boundary. (O-Q) Delayed and aberrant SA extension (visualized using anti-Tuj1 antibody) in the
absence of MEs upon Hb9
MN
::Cre/PGKneolox2DTA co-transfection. (R) Longitudinal analysis of relative SA and ME extension, and impact of ME removal
(n7 sections, 3 embryos per stage and condition). (S) Peripheral axons visualized in E10.5 mouse embryo. (T-W) SAs, labeled using the Brn3a
tau:lacZ
allele, extending along preformed MEs, labeled using the Hb9
MN
::GFP transgene. (X-Z) Delayed and aberrant SA extension in the absence of MEs in
Olig2
Cre
;Rosa26
lxstopDTA
embryos. (AA) Longitudinal analysis of relative SA and ME extension, and impact of ME removal (n8 sections, 4 embryos per stage
andcondition). hpf, hourspost-fertilization; DRG, dorsal root ganglion; MN, motor neurons. Error bars indicatestandarderror of themean(s.e.m.). Scalebars: 10 minE,
H for B-H; 50 m in K-Q,T-Z.
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extension, however, impacts on the establishment of peripheral SA
trajectories were considerably milder in both chick (Fig. 2A-C) and
mouse (Fig. 2D-G), resembling results obtained by previous studies
relying on late-stage surgical or incomplete genetic removal of motor
neurons (Huettl et al., 2011; Wang and Scott, 1999; Wenner and
Frank, 1995).
Sensory axons are invariably guided by motor axons
Do the trajectories chosen by MEs therefore serve as a template for
the assembly of peripheral nerves by guiding later-extending axon
types? We further tested this by genetically forcing MEs to choose
aberrant trajectories, followed by visualizing SAs. In zebrafish, this
was achieved by injecting morpholino oligonucleotides targeting a
splice variant of the MUSK (muscle-specific kinase receptor) gene
that were previously established to selectively alter the pattern of
ME extension (Zhang et al., 2004). In these larvae, SAs faithfully
recapitulated aberrant trajectory choices made by MEs (Fig. 3A-I),
with SAs invariably skipping trajectories not occupied by MEs
(Fig. 3J,K). In parallel, we took advantage of two previously
established gene disruptions in mouse that cause varying degrees of
ME misrouting at a binary axon choice point at the base of the limb
(supplementary material Fig. S4A-H) (Helmbacher et al., 2000;
Kramer et al., 2006; Luria et al., 2008). First, the motor neuron-
restricted expression of the receptor tyrosine kinase (RTK) EphA4
was selectively eliminated in the motor neuron lineage through
Cre/loxP recombination in Epha4
fx/fx
;Olig2
Cre
embryos. Second,
the expression of the RTK Ret, which is largely motor neuron
restricted at the relevant embryonic stages (E9.5-E12), was
eliminated in Ret
/
embryos. Both models selectively affected
the dorsal choice of MEs originating from the lateral division of the
lateral motor column by altering their responsiveness towards
mesenchymal guidance cues (Fig. 3M,O,Q,S,U; supplementary
material Fig. S4I-Q) (Helmbacher et al., 2000; Kramer et al., 2006;
Luria et al., 2008). As in zebrafish, aberrant ME projections were
closely mirrored by SAprojections in the mouse hindlimb (Fig. 3L-V;
supplementary material Fig. S4I-Q) and revealed a linear
relationship between the extent of ME projections and that of the
later-extending SAs (Fig. 3W). In both models, SAs thus continued
to tightly adhere to MEs, indicating a restriction of EphA4 and Ret
function to ME-mesenchyme signaling, but not in SA-ME
interactions, during limb innervation. Taken together, SAs thus
appear to invariably favor and tightly adhere to trajectories occupied
by earlier extending MEs.
Sensory axons are dispensable for motor axon guidance
We next asked whether these data reflected a hierarchical axon type-
dependent relationship by testing whether SAs would conversely
influence ME extension by DTA-mediated genetic ablation of DRG
neurons in mouse embryos (supplementary material Fig. S5A-F).
Consistent with previous data (Huettl et al., 2011), genetic removal of
DRG neurons in mouse led to defasciculation of MEs (Fig. 4A,B),
which could reflect the loss of repulsive activities exerted by SAs on
MEs (Gallarda et al., 2008). As the absence DRG sensory neurons
prevents the assembly of DRGs proper, which in turn provide a
niche for the expansion of Schwann cell precursors, the
defasciculated appearance of MEs could have been alternatively
(or additionally) caused by reduced numbers of Schwann cell
precursors. At the same time, absence of SAs did not influence the
accuracy of trajectory or target selection by MEs (Fig. 4C-F),
consistent with MEs normally extending ahead of SAs. SAs thus
appear to invariably depend on pre-extending MEs for establishing
normally patterned peripheral trajectories, but not vice versa,
whereas SAs exert repulsive activities that prevent aberrant
intermingling with (and possibly defasciculation of ) MEs.
Fig. 2. Effects of partial MEablation on SAextension. (A) Dorsal
whole-mount view of lumbar spinal cord and limbs in E6 chick
embryo: peripheral axons visualized by anti-Tuj1 immunodetection
(black). Severe reduction, but not loss, of crural (asterisk),
peroneal (PN) and tibial nerves (TN) (arrowheads) upon unilateral
transfection with a low titer (0.5 g/ml) of Hb9
MN
::Cre/
PGKneolox2DTA plasmids. LFC, lateral femoral cutaneous nerve.
(B,C) Transverse section of E6 chick spinal cord: partial ablation of
motor neurons (MNs) after unilateral low-titer transfection. Anti-Isl1/2
immunofluorescence (gray) to label DRG neurons and MNs.
Anti-TrkA immunofluorescence (yellow) to label DRG neurons.
(D) Dorsal whole-mount viewof SAs (yellowindicates Brn3a
tlz
) at the
sciatic plexus (SP) in E12.5 mouse embryo. DPN, deep peroneal
nerve; PN, peroneal nerve; TN, tibial nerve. (E) Reduction, but not
loss, of SAs beyond the sciatic plexus after delayed ablation of MEs
in Olig2
Cre
;Isl2
lxstopDTA
embryo. (F) Transverse section of E12.5
control spinal cord: MNs labeled using Hb9
MN
::GFP(magenta). Anti-
Isl1/2 immunofluorescence (gray) visualizes nuclei of DRG neurons
and MNs. (G) Transverse section of E12.5 Olig2
Cre
;Isl2
lxstopDTA
spinal cord: severe reduction, but not complete absence, of MNs.
Scale bars: 300 m in A; 100 m in B; 200 m in D,F.
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Sympathetic axon trajectories are configured by
pre-extending sensory and motor axons
Consistent with previous observations (Anet al., 2002; Coughlin et al.,
1977; Yip, 1990), sympathetic efferent axons (SEs) emerging from
sympathetic chain ganglia (SCGs), visualized by tyrosine hydroxylase
(TH) immunodetection, were the last axons to extend peripherally in
zebrafish (Fig. 5A-E), chick (Fig. 5F-L) and mouse (Fig. 5M-Q), thus
recapitulating the phylogenetically late innervation of neural crest-
derived autonomic circuits (Butler and Hodos, 2005; Holland, 2009).
At trunk levels, SEs emerging fromSCGs followthree principal routes
to access effector organs (Elfvin, 1983): (1) a minor medial-visceral
route; (2) a longitudinal route along sympathetic chain or arteries;
and (3) a lateral route adhering to the initial trajectories of MEs and
SAs (Fig. 5A,L,M,Q), running parallel to, but not overlapping with,
intersegmental blood vessels (Fig. 6A-C; supplementary material
Fig. S6E-J) (Nakao and Ishizawa, 1994). In amniotes, the lateral
route SEs eventually project along pre-extending cutaneous SAs
to innervate dermal glands and smooth muscle as part of the
circuits underlying skin thermoregulation (Figs 6D-J and 7C,D)
(Elfvin, 1983; Nakao and Ishizawa, 1994).
We next tested whether this sequential extension pattern reflected
a dependence of SEs on preformed SAs and/or MEs in mouse
Fig. 3. SA trajectories are configured by pre-extending MEs. (A-I) RoP and CaP SA (yellow) and ME (magenta) trajectories in 72 hpf control zebrafish
embryos (A-C) and upon unp-SV1-MO morpholino injection (D-I). (A,B) Same principal trajectories followed by SAs and MEs in control embryo.
(C) Reconstructed main ME and SA trajectories in control (staggered rendering of MEs and SAs for simplicity). (D-I) Examples of SAs (D,G) following
aberrant ME trajectories, skipping trajectories devoid of MEs (E,H) in unp-SV1-MO-injected embryos. (F,I) Reconstructed main ME and SA trajectories of the
same embryos. (J,K) Cumulative semi-quantification: ratio of nerve segments with SAs (J) or MEs (K) possessing (yes) or lacking (no) MEs (J) or SAs (K),
respectively, in control or unp-SV1-MO-injected embryos at individual nerve segments (control: n=12 segments, 6 embryos; unp-SV1-MO injection: n=9
segments, 5 embryos). (L-Q) Dorsal whole-mount views of SAs (L,N,P) and MEs (M,O,Q) in peroneal nerve (PN) in E12.5 mouse embryo lacking EphA4 in
the motor neuron lineage (Epha4
cko
): extent of ME projection defects (classified as ranging from I-III) are mirrored by SAs. (R-U) Transverse sections of the
sciatic plexus in E12.5 control (R,S) and Ret-deficient (Ret
ko
) (T,U) mouse embryos: SA (T) projections mirror severely reduced/absent dorsal ME projections
(U) in Ret
ko
(asterisk). PN, peroneal nerve; TN, tibial nerve. (V) Correlation of SA and ME extension in mutants with varying ME projection defects at sciatic
plexus (mut I-III). (W) Plotting SA against ME pn diameters reveals a linear relationship between extent of SA and ME extension in control, Epha4
cko
and
Ret
ko
embryos (n=9, 16 and 17: control, Epha4
cko
and Ret
ko
embryos, respectively). Scale bars: 20 m in B,E,H; 200 m in M,O,Q; 100 m in S,U.
Fig. 4. SAs influence ME fasciculation but not trajectory
or target choice. (A,B) Dorsal whole-mount view: MEs
extending into hindlimbs in control (A) and in the absence
of SAs in Wnt1
Cre
;Rosa26
lxstopDTA
mouse embryos (B).
CP, crural plexus; DPN, deep peroneal nerve; PN, peroneal
nerve; SP, sciatic plexus; TN, tibial nerve. (C-F) Transverse
section of E12.5 thoracic (C,D) and lumbar (E,F) motor
columns (magenta indicates Hb9
MN
::GFP): retrograde DiI
tracing does not detect aberrant ME targeting at the level of
columnar divisions. (C) Retrograde DiI tracing from epaxial
muscle labels medial division of medial motor column (MMC).
(D) Retrograde DiI tracing from hypaxial muscle labels lateral
MMC. (E) Retrograde DiI tracing from ventral hindlimb labels
Hb9::eGFP
low
medial division of lateral motor column (LMC).
(F) Retrograde DiI tracing from dorsal hindlimb labels
Hb9
MN
::GFP
high
lateral LMC (asterisk indicates a possible
Hb9
MN
::GFP
high
LMCl neuron in the process of lateral
migration). Scale bars: 300 m in A,B; 50 m in C-F.
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models that allowed us to separately address the contribution of each
of the three principal axon types to peripheral nerve assembly
(supplementary material Fig. S6A-D). This was achieved by
separately interbreeding the Cre recombination-controlled DTA
expression lines R26
fxDTA
(Ivanova et al., 2005) or Isl2
lxstopDTA
(Yang et al., 2001) with previously established mouse lines that
drive Cre expression in motor neuron progenitors (Ivanova et al.,
2005), DRG neurons (Hasegawa et al., 2007) or SCG neurons
(Parlato et al., 2007) (supplementary material Fig. S6A-D). We
found that the aberrant cutaneous SA projection patterns resulting
from the absence of MEs (Wang et al., 2011) were precisely
mirrored by SEs (Fig. 7F,G), including frequent failure of dermal
target innervation by both SEs and SAs (Fig. 7H,I). This suggested
that MEs are dispensable for initiating the peripheral extension of
SEs, but indirectly influence their trajectory by determining the
pattern of SA projections (Fig. 7J). We further tested this by
studying SE projections upon selective genetic removal of SAs. In
the absence of SAs, SEs extended peripherally along motor
projections (Fig. 7K,L), but consistently failed to enter cutaneous
trajectories, leaving the trunk dermis entirely devoid of innervation
(Fig. 7M-O). At the same time, intersegmental blood vessels
developed normally in the absence of MEs and SAs (supplementary
material Fig. S6E-J), indicating that failure of cutaneous SE
projections upon SA or ME removal was not indirectly caused by
impacts on vascular patterning. Last, selective genetic removal of
SEs did not result in detectable alterations of peripheral ME or SA
projections (Fig. 7P-T), consistent with the initiation of SE
extension after most ME and SA trajectories have been
established. Thus, although a subset of SEs uses the developing
vasculature to access peripheral end organs (Makita et al., 2008),
trunk cutaneous SE projections are absolutely reliant on their
association with pre-formed SAtrajectories that, in turn, are initially
guided by MEs (Fig. 8A).
DISCUSSION
Our data uncover a conserved hierarchy of axon type-dependent
extension and selective fasciculation events governing vertebrate
peripheral nerve assembly (Fig. 8A), the temporal order of which
recapitulates the successive phylogenetic emergence of peripheral
axon types and circuits in the vertebrate lineage (Fig. 8B-F). First,
MEs actively navigate to skeletal muscle targets guided by
mesenchymal cues (Bonanomi and Pfaff, 2010; Dasen, 2009),
thus establishing an initial grid of peripheral trajectories that
provides a template for subsequent peripheral nerve assembly
(Fig. 8B). Second, SAs use preformed ME trajectories as gateways
to their peripheral target organs (Fig. 8C-E). Third, subsets of SEs
eventually follow these pre-established trajectories, presumably by
responding to cues on SAs (Fig. 8D-F).
MEs extending from cholinergic motor neurons to muscles
involved in locomotion represent the sole common feature of
peripheral nerves in extant chordates, predating both vertebrates and
neural crest-derived circuits (Fig. 8B) (Candiani et al., 2012; Denes
et al., 2007; Fetcho, 1992). SAs and SEs, which emerged in
agnathostomes and gnathostomes, respectively (Butler and Hodos,
2005; Holland, 2009), were thus from the outset able to rely on pre-
evolved axon pathways for accessing peripheral end organs
(Fig. 8C-F), This pattern could have been enforced by the
segmental remodeling in gnathostomes that brought ventral and
dorsal peripheral nerve roots in closer apposition (Butler and Hodos,
2005; Jorgensen, 1998).
The timing of axon extension can profoundly influence the
outcome of heterotypic axon-axon encounters (Wang and Marquardt,
2013), which may have been a key factor in preserving peripheral
axon type hierarchies from fish to mouse. For example, at least in
mouse, the early extension of MEs seems to promote selective
fasciculation of SAs with pre-extending MEs by encouraging reverse
activation of ephrin A proteins on SA growth cones by their cognate
EphA RTKs located on MEs (Wang et al., 2011). In addition, this
configuration effectively discourages illicit forward activation of
repulsive signaling by the same EphAs on ME growth cones by their
cognate ephrin A interaction partners on SAs (Gallarda et al., 2008;
Wang et al., 2011), which could otherwise jeopardize the role of MEs
in pioneering peripheral nerve tracts. The retention of this hierarchy of
axon type-dependent interactions could have been further promoted
by the prioritized assembly of ME and RB-based escape reflex arcs
Fig. 5. Conserved late extension of sympathetic efferent axons (SEs).
(A) Transverse section of 32 dpf zebrafish: SEs [blue, anti-tyrosine
hydroxylase (TH) immunofluorescence] extending from sympathetic chain
ganglion (SCG) along preformed peripheral nerves (red, SA/ME: anti-Tuj1
immunofluorescence). (B,C) TH
+
SCG neurons prior to initiation SE axon
extension at 18 dpf. (D,E) Higher magnifications of A: TH
+
SEs (arrowheads)
extending along preformed peripheral axons. (F) Transverse sections of E7
chick embryo at trunk levels: TH
+
SCG neurons (blue) around initiation SE
extension relative to preformed PNs (red). (G,H) Magnified view of SCG
neurons relative to preformed peripheral nerves. (I,J) TH
+
SE axons begin
extending from SCG along PNs at E8 (arrowheads). (K,L) SE axon
advancing further peripherally along peripheral nerves at E9 (arrowheads).
(M) Transverse section of E12.5 mouse embryo: TH
+
SEs (arrowhead)
beginning to extend fromSCGs along pre-extending MEs and SAs in the ramus
communicans (RC). (N,O) SCG and rc just prior to initiation of SE extension
(arrowhead). (P,Q) Detailed view of SEs (arrowheads) extending along SAs
and MEs of RC at E12.5. nc, notochord; DR/VR, dorsal/ventral ramus; vi,
intersegmental blood vessel; DNR/VNR, dorsal/ventral nerve roots; RC, ramus
communicans. Scale bars: 20 m in A,C,E; 100 m in F,H,J,L,M; 50 m in O,Q.
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over more advanced SAand SE-based somatosensory and autonomic
circuits in anamniotes developing through pelagic larval stages
(Fig. 8B,C).
Resolving axon type-dependent relationships in the context of the
embryo remains challenging, because the underlying signaling
mechanisms may operate independently from those determining the
responsiveness of axons towards non-axonal guidance cues (Raper
and Mason, 2010; Wang and Marquardt, 2013). Thus, altering
axon-axon signaling tends to produce changes of axonal trajectories
within the confines of relatively fixed pathways that are determined
by tissue tracts that are permissive or non-permissive to axon
growth. Advancing our understanding of the contribution of axon-
axon interactions to nervous system development will therefore
depend on expanding the toolkit for unambiguously distinguishing
or manipulating the different axon types involved.
Although some of the ontophyletic considerations put forward
here presently remain speculative, our findings pave the way for
systematically exploring the cellular and molecular basis of the
axon-axon interactions contributing to peripheral nerve assembly,
and may ultimately serve as a generalized model for how, during
nervous system development, phylogenetically newer and older
neuron types assemble into common circuitries.
MATERIALS AND METHODS
Zebrafish
To label MEs, the previously established transgenic zebrafish line Tg(NBT:
dsRed) was used, employing a genomic fragment of the Xenopus laevis gene
neuronal beta-tubulin (NBT) containing motor neuron regulatory modules to
drive red fluorescent protein (dsRed) expression (Blader et al., 2003). To label
SAs, the line Tg(-8.4neurog1:GFP) was used, which takes advantage of a
genomic fragment of the neurogenin 1 gene employing DRGneuron-restricted
regulatory elements to drive green fluorescent protein (GFP) expression
(Peri and Nusslein-Volhard, 2008). To prevent MEextension, two morpholino
oligonucleotides (MO) designed against the Islet1 mRNA [islet1E2-MO
(5
0
-TTAATCTGCGTTACCTGATGTAGTC-3
0
) and islet1E3-MO (5
0
-GAAT
GCAATGCCTACCTGCCATTTG-3
0
)] were co-injected that have previously
been observed to interfere selectively with motor neurogenesis (Hutchinson
and Eisen, 2006). To alter, but not prevent, ME extension, we used an MO
against the splice variant 1 (SV1) of muscle-specific kinase (MuSK) (unp-
SV1-MO: 5
0
-TATTGTCTTACCTCCATTCTACGGG-3
0
) that has previously
been observed to trigger altered MEnavigation (Zhang et al., 2004). MOs were
injected at one- to two-cell stage with the following amounts: islet1E2-MO,
3 ng; islet1E3-MO, 3 ng; and unp-SV1-MO, 8 ng. All MOs were obtained
from Gene Tools (Philomath, OR, USA).
Chick
Fertilized eggs were staged according to Hamburger and Hamilton (HH)
(Hamburger and Hamilton, 1992) and transfected using plasmid injection
into the neural tube and in ovo electroporation at HH 12/13 as previously
described (Marquardt et al., 2005). To label MEs, a previously characterized
4.5 kb promoter fragment of the Hb9 gene, Hb9
MN
, containing motor
neuron-specific regulatory elements (Lee et al., 2004) was fused to mCherry
(Cherry red fluorescent protein with a myristoylation signal m for
membrane tethering). To co-label SAs, a previously characterized DRG-
specific regulatory module of the Isl1 gene, Isl1
DRG
, was fused to a minimal
TATA box and mGFP (m-green fluorescent protein) (Uemura et al., 2005). In
addition to DRG neurons, Isl1
DRG
labels a subset of Schwann cell
precursors (SCPs), presumably originating from their proliferation niche in
the DRG(e.g. Fig. 1N, asterisks). For imaging, transverse sections were thus
selected containing fewer SCPs to allow unimpeded visualization of SAs.
To prevent ME extension, motor neurons were ablated by co-injecting
PGKneolox2DTA (Addgene: Plasmid 13449) with Hb9
MN
::Cre for
Cre-mediated activation of DTA expression in motor neurons. After
electroporation, eggs were incubated at 38C until the desired stages.
Mouse
All mouse work conformed to regulations by the University Medical
Center Gttingen animal welfare committee and German animal welfare
laws. MEs and SAs were co-labeled by interbreeding the previously
established motor neuron-specific marker line Hb9
MN
::GFP (Lee et al.,
2004) and DRG-neuron marker line Brn3a
tau:lacZ
(Trieu et al., 2003). For
preventing ME extension, the Cre-controlled ubiquitously expressed
diphtheria toxin (DTA) expression line Rosa26
lxstopDTA
(Jax stock
#006331) was interbred with the motor neuron progenitor ( pMN)-
specific Cre-driver line Olig2
Cre
to selectively ablate pMNs (Ivanova et al.,
2005). For preventing SA extension, the neural crest-restricted Cre-deleter
Fig. 6. SEs extend along MEs and SAs to innervate dermis.
(A-C) Transverse section of E14.5 mouse embryo at trunk levels: the
majority of trunk-innervating SEs (arrowheads) extend along
pre-extending axons (magenta indicates Hb9::eGFP) (A,C), rather
than intersegmental blood vessels (vi) (gray indicates anti-Pecam1
immunofluorescence) (B,C). Subsets of SEs directly extend medially
(m) and dorsally (d) along vasculature (B,C). (D-F) Dorsal whole-
mount view of E18.5 mouse: dorsal cutaneous nerve (dCN) axons
fanning out in trunk dermis and longitudinal projections by MEs into
subdermal cutaneous maximus (cm) muscle (D,F). (E) SE projections
(blue indicates anti-TH immunofluorescence) through dCN into
dermis. (F) ME (magenta indicates Hb9::eGFP) innervation of
subdermal cm muscle. (G-I) Longitudinal section through dCN at
E18.5: separately labeled SEs (blue) and SAs (yellow indicates
anti-TrkA immunofluorescence) can be seen. (J) Three axon types in
dorsal ramus (ventral ramus, visceral or vascular trajectories are not
depicted for simplicity). Magenta dots indicate cross-sections of cm
MEs. DR, dorsal ramus; vi, intersegmental blood vessel; RC, ramus
communicans; VR, ventral ramus. Scale bars: 150 m in A; 300 m in
D; 50 m in G.
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line Wnt1
Cre
(Jax stock #009107) (Hayashi and McMahon, 2002) was
interbred with the Cre-controlled diphtheria toxin (DTA) expression line
Isl2
lxstopDTA
(Jax stock #007942) (Yang et al., 2001), thus leading to
ablation of Isl2
+
DRG neurons derived from Wnt1
+
neural crest cells, but
not Isl2
+
motor neurons. To study impacts on SEs, and to prevent potential
ablation of Isl2
+
SCG neurons, the DRG neuron-restricted Cre line
Advillin
Cre
(Hasegawa et al., 2007) was interbred with Isl2
lxstopDTA
. SE
extension was prevented by interbreeding DBH
Cre
(Parlato et al., 2007)
with Rosa26
lxstopDTA
. To selectively misroute MEs in the hindlimb,
homozygous Epha4
flox
(Herrmann et al., 2010) mice were interbred with
Fig. 8. Conserved axon type hierarchy and ontophyletic model of peripheral nerve assembly. (A) Hierarchical relationships between the three principal
peripheral axon types (whether MEs directly or indirectly influence SEs remains unresolved). (B-F) Ontophyletic model of PN assembly. (B) The phylogenetically
oldest MEs extend from MNs in neural tube and actively navigate to peripheral targets guided by mesenchymal cues. RBs contribute to cutaneous escape
reflexes prior to emergence of neural crest-derived SAs. (C) SAs use preformed ME trajectories as gateways to peripheral targets. (D) Neural crest-derived SEs
subsequently extend peripherally along trajectories established by SAs and MEs. (E) Cutaneous SAs eventually project beyond MEs to innervate dermis,
followed by SEs. RBs are lost at the transition to the amniote lineage or degenerate in adult gnathostome anamniotes. (F) Principal pattern of trunk peripheral axon
types in a prototypical gnathostome (VR, visceral or vascular trajectories are not depicted for simplicity).
Fig. 7. SE trajectories are configured by pre-extending SAs and MEs. (A,B) Transverse section of E14.5 mouse embryo: SE (blue), SA (yellow) and ME
(magenta) axons extending into dorsal (DR) and ventral (VR) nerve rami. (C) Whole-mount view of dorsal cutaneous nerve (dCN) axons fanning out into trunk
dermis. (D) Visualization of SE axons only in same specimen. (E) Summary: three axon types in DR (VR, visceral or vascular trajectories are not depicted for
simplicity). Magenta dots indicate cross-sectioned longitudinally projecting cutaneous maximus (cm) MEs. (F,G) Loss of dorsal (asterisk) and ventral misrouting of
SA projections in the absence of MEs (Olig
Cre
;Rosa26
fxstopDTA
) (F) mirrored by SE (G) (asterisk). The converse dorsal misrouting of ventral SAs observed upon ME
removal is not shown for simplicity. (H,I) Intermittent loss of dCNs (asterisks) and aberrant pattern of SA projections in the absence of MEs is mirrored by SEs (I).
(J) Summary of F-I. (K-N) Initial peripheral extension of SEs along MEs in the absence of SAs (Adv
Cre
;Isl2
fxstopDTA
) (note the higher degree of SE fasciculation,
compared with control), but failure of SEs to innervate dermis (M,N) (remaining axons in Mare subdermal cmMEs). (O) Summary of K-N. (P-Q) Normal appearance
of ME, SAtrajectories in the absence of SEs (Dbh
Cre
;Rosa26
fxstopDTA
). (R,S) Normal appearance of dorsal cutaneous nerves in the absence of SEs. (T) Summary of
appearance of P-S. All images are representative of at least five embryos per condition. Scale bars: 100 min B,G,L,Q; 300 min D,I,N,S. sg, sympathetic ganglion.
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Olig2
Cre
to specifically inactivate Epha4 in pMNs or embryos homozygous
for a Ret-null allele (Ret
ko
) (Schuchardt et al., 1994) were studied. The
mouse lines were genotyped as described previously.
Immunohistochemistry and imaging
Immunofluorescence staining was performed as described previously (Wang
et al., 2011). Primary antibodies used were: rabbit anti--galactosidase
(Cappel, 1:6000; 55976); goat anti--galactosidase (Cappel, 1:12,000;
56028); rabbit anti-GFP (Molecular Probes, 1:2000; AB11122); sheep anti-
GFP (Biogenesis, 1:6000; 4745-1051); chicken anti-GFP (Abcam, 1:3000;
AB13970); Rabbit anti-dsRed (Clontech, 1:1000; 632496); mouse anti-Tuj-1/
III-tubulin (Abcam, 1:3000; MMS-435P); rabbit anti-TH (Millipore,
1:1500; 657012); goat anti-TH (Millipore, 1:1500; AB1542); biotin rat
anti-mouse PECAM-1 (BD Pharmingen, 1:3000; 553371); rabbit anti-Isl1/2
(gifts from S. L. Pfaff, Salk Institute, USA; 1:3000); Isl1/2 (DSHB, 39.4D5,
1:200); rabbit anti-TrkA (a gift from L. F. Reichardt, UCSF, USA; 1:1000);
neurofilament (DSHB, 2H3, 1:200) and neurofilament (DSHB, 4H6, 1:200).
Immunofluorescence was detected with Alexa-488, -555 and -637, and
streptavidin conjugated secondary antibodies (Molecular Probes, all at
1:1000). Images were collected using Zeiss (LSM 710) or Leica TCS/MP
confocal/two-photon microscopes.
Retrograde tracing
Hb9::eGFPtransgenic mouse embryos were used to identify motor trajectories.
DiI (Sigma)-labeled embryos were incubated in 4% paraformaldehyde
overnight at 37C to permit diffusion, prior to vibratome sectioning at
120 m for analysis.
Live imaging of zebrafish
Injected embryos were dechorionated, anesthetized in 0.004% tricaine and
mounted in 0.8% low melting agarose on 35 mm glass-bottom dishes. Live
imaging was performed at 28C using a LSM5 Live confocal microscope
(Carl Zeiss Microimaging).
Quantifying the relationship between motor axon and SA
extension
For zebrafish larvae, two segments from each were randomly selected for
quantification. For each segment, the main branches of motor axon and
SAaxons were classified in two categories: yes, indicating motor axon or SA
axons project in close association with their counterparts; and no, indicating
motor axon or SA axons extend independently. Next, the percentages of yes
and no among total incidences were calculated for motor axon and SA,
respectively. In mouse embryos, the diameters of all motor axons and SAs in
peroneal nerves were separately measured at midsection for each embryo.
Acknowledgements
We thank A. Klusowski and B. Veith for outstanding technical and administrative
support, D. Mu ller for technical advice, G. Schu tz for providing Dbh
Cre
mice,
U. Stra hle for providing the -8.4neurogenin:GFP line, B. Novitch and T. M. Jessell for
providing Olig2
Cre
mice, and E. Turner for Brn3a
tau:lacZ
mice.
Competing interests
The authors declare no competing financial interests.
Author contributions
L.W. and T.M. devised the project and designed experiments. A.M. and C.N.-V.
performed all zebrafish experiments. L.W. performed all experiments on mouse and
chick embryos, apart from analysis of Ret
ko
embryos by D.B. and S.L.P. L.C.
designed vectors for SAtracing and ME ablation in chick. T.M. wrote the manuscript.
Funding
This work was funded by a grant of the Deutsche Forschungsgemeinschaft (DFG)
(MA4278/4-1), the Emmy Noether Program of the DFG (MA4278/1-1) and the
Go ttingen center of excellence for nanomicroscopy and cellular physiology of the brain
(CNMPB). The ENI-Gis a cooperative of the University Medical Center Go ttingen and
the Max-Planck Gesellschaft. Deposited in PMC for immediate release.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.106211/-/DC1
References
An, M., Luo, R. and Henion, P. D. (2002). Differentiation and maturation of zebrafish
dorsal root and sympathetic ganglion neurons. J. Comp. Neurol. 446, 267-275.
Blader, P., Plessy, C. and Stra hle, U. (2003). Multiple regulatory elements with
spatially and temporally distinct activities control neurogenin1 expression in
primary neurons of the zebrafish embryo. Mech. Dev. 120, 211-218.
Bonanomi, D. and Pfaff, S. L. (2010). Motor axon pathfinding. Cold Spring Harb.
Perspect. Biol. 2, a001735.
Butler, A. B. and Hodos, W. (2005). Comparative Vertebrate Neuroanatomy:
Evolution and Adaptation, 2nd edn. London, UK: J.C. Wiley & Sons.
Candiani, S., Moronti, L., Ramoino, P., Schubert, M. and Pestarino, M.
(2012). A neurochemical map of the developing amphioxus nervous system.
BMC Neurosci. 13, 59.
Coughlin, M. D., Boyer, D. M. and Black, I. B. (1977). Embryologic development of
a mouse sympathetic ganglion in vivo and in vitro. Proc. Natl. Acad. Sci. U.S.A. 74,
3438-3442.
Dasen, J. S. (2009). Transcriptional networks in the early development of sensory-
motor circuits. Curr. Top. Dev. Biol. 87, 119-148.
Denes, A. S., Je kely, G., Steinmetz, P. R. H., Raible, F., Snyman, H.,
Prudhomme, B., Ferrier, D. E. K., Balavoine, G. and Arendt, D. (2007).
Molecular architecture of annelid nerve cord supports common origin of
nervous system centralization in bilateria. Cell 129, 277-288.
Elfvin, L. (1983). Autonomic Ganglia. London, UK: John Wiley & Sons.
Fetcho, J. R. (1992). The spinal motor system in early vertebrates and some of its
evolutionary changes. Brain Behav. Evol. 40, 82-97.
Gallarda, B. W., Bonanomi, D., Muller, D., Brown, A., Alaynick, W. A.,
Andrews, S. E., Lemke, G., Pfaff, S. L. and Marquardt, T. (2008). Segregation
of axial motor and sensory pathways via heterotypic trans-axonal signaling.
Science 320, 233-236.
Hamburger, V. (1929). Experimentelle Beitra ge zur Entwicklungsphysiologie der
Nervenbahnen in der Froschextremita t. Rouxs Arch. Dev. Biol. 119, 47-99.
Hamburger, V. and Hamilton, H. L. (1992). A series of normal stages in the
development of the chick embryo. Dev. Dyn. 195, 231-272.
Hasegawa, H., Abbott, S., Han, B.-X., Qi, Y. and Wang, F. (2007). Analyzing
somatosensory axon projections with the sensory neuron-specific Advillin gene.
J. Neurosci. 27, 14404-14414.
Hayashi, S. and McMahon, A. P. (2002). Efficient recombination in diverse tissues
by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene
activation/inactivation in the mouse. Dev. Biol. 244, 305-318.
Helmbacher, F., Schneider-Maunoury, S., Topilko, P., Tiret, L. and Charnay, P.
(2000). Targeting of the EphA4 tyrosine kinase receptor affects dorsal/ventral
pathfinding of limb motor axons. Development 127, 3313-3324.
Herrmann, J. E., Pence, M. A., Shapera, E. A., Shah, R. R., Geoffroy, C. G. and
Zheng, B. (2010). Generation of an EphA4 conditional allele in mice. Genesis 48,
101-105.
Holland, L. Z. (2009). Chordate roots of the vertebrate nervous system: expanding
the molecular toolkit. Nat. Rev. Neurosci. 10, 736-746.
Honig, M. G., Lance-Jones, C. and Landmesser, L. (1986). The development of
sensory projection patterns in embryonic chick hindlimb under experimental
conditions. Dev. Biol. 118, 532-548.
Honig, M. G., Frase, P. A. and Camilli, S. J. (1998). The spatial relationships
among cutaneous, muscle sensory and motoneuron axons during development of
the chick hindlimb. Development 125, 995-1004.
Huettl, R.-E., Soellner, H., Bianchi, E., Novitch, B. G. and Huber, A. B. (2011).
Npn-1 contributes to axon-axon interactions that differentially control sensory and
motor innervation of the limb. PLoS Biol. 9, e1001020.
Hutchinson, S. A. and Eisen, J. S. (2006). Islet1 and Islet2 have equivalent abilities
to promote motoneuron formation and to specify motoneuron subtype identity.
Development 133, 2137-2147.
Ivanova, A., Signore, M., Caro, N., Greene, N. D. E., Copp, A. J. and
Martinez-Barbera, J. P. (2005). In vivo genetic ablation by Cre-mediated
expression of diphtheria toxin fragment A. Genesis 43, 129-135.
Jorgensen, J. M. (1998). The Biology of Hagfishes, 1st edn. London: Chapman and
Hall, Thomson Science.
Katz, M. J. (1983). Ontophyletics: studying evolution beyond the genome. Perspect.
Biol. Med. 26, 323-335.
Katz, M. J., Lasek, R. J. and Silver, J. (1983). Ontophyletics of the nervous system:
development of the corpus callosum and evolution of axon tracts. Proc. Natl.
Acad. Sci. U.S.A. 80, 5936-5940.
Kramer, E. R., Knott, L., Su, F., Dessaud, E., Krull, C. E., Helmbacher, F. and
Klein, R. (2006). Cooperation between GDNF/Ret and ephrinA/EphA4 signals for
motor-axon pathway selection in the limb. Neuron 50, 35-47.
Lee, S.-K., Jurata, L. W., Funahashi, J., Ruiz, E. C. and Pfaff, S. L. (2004).
Analysis of embryonic motoneuron gene regulation: derepression of general
activators function in concert with enhancer factors. Development 131,
3295-3306.
Luria, V., Krawchuk, D., Jessell, T. M., Laufer, E. and Kania, A. (2008).
Specification of motor axon trajectory by ephrin-B:EphB signaling: symmetrical
control of axonal patterning in the developing limb. Neuron 60, 1039-1053.
1882
RESEARCH ARTICLE Development (2014) 141, 1875-1883 doi:10.1242/dev.106211
D
E
V
E
L
O
P
M
E
N
T
Ma, L. H., Gilland, E., Bass, A. H. and Baker, R. (2010). Ancestry of motor
innervation to pectoral fin and forelimb. Nat. Commun. 1, 49.
Makita, T., Sucov, H. M., Gariepy, C. E., Yanagisawa, M. and Ginty, D. D. (2008).
Endothelins are vascular-derived axonal guidance cues for developing
sympathetic neurons. Nature 452, 759-763.
Marquardt, T., Shirasaki, R., Ghosh, S., Andrews, S. E., Carter, N., Hunter, T.
and Pfaff, S. L. (2005). Coexpressed EphA receptors and ephrin-A ligands
mediate opposing actions on growth cone navigation from distinct membrane
domains. Cell 121, 127-139.
Nakao, T. and Ishizawa, A. (1994). Development of the spinal nerves in the mouse
with special reference to innervation of the axial musculature. Anat. Embryol.
(Berl) 189, 115-138.
Parlato, R., Otto, C., Begus, Y., Stotz, S. and Schutz, G. (2007). Specific ablation
of the transcription factor CREB in sympathetic neurons surprisingly protects
against developmentally regulated apoptosis. Development 134, 1663-1670.
Peri, F. and Nu sslein-Volhard, C. (2008). Live imaging of neuronal degradation by
microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell 133,
916-927.
Raper, J. and Mason, C. (2010). Cellular strategies of axonal pathfinding. Cold
Spring Harb. Perspect. Biol. 2, a001933.
Schuchardt, A., DAgati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V.
(1994). Defects in the kidney and enteric nervous system of mice lacking the
tyrosine kinase receptor Ret. Nature 367, 380-383.
Spitzer, N. C. (1984). What do Rohon-Beard cells do? Trends Neurosci. 7, 224-225.
Swanson, G. J. and Lewis, J. (1986). Sensory nerve routes in chick wing buds
deprived of motor innervation. J. Embryol. Exp. Morphol. 95, 37-52.
Taylor, A. C. (1944). Selectivity of nerve fibers from the dorsal and ventral roots in
the development of the frog limb. J. Exp. Zool. 96, 159-185.
Trieu, M., Ma, A., Eng, S. R., Fedtsova, N. and Turner, E. E. (2003). Direct
autoregulation and gene dosage compensation by POU-domain transcription
factor Brn3a. Development 130, 111-121.
Uemura, O., Okada, Y., Ando, H., Guedj, M., Higashijima, S.-i., Shimazaki, T.,
Chino, N., Okano, H. and Okamoto, H. (2005). Comparative functional genomics
revealed conservation and diversification of three enhancers of the isl1 gene for
motor and sensory neuron-specific expression. Dev. Biol. 278, 587-606.
Wang, L. and Marquardt, T. (2013). What axons tell each other: axon-axon
signaling in nerve and circuit assembly. Curr. Opin. Neurobiol. 23, 974-982.
Wang, G. and Scott, S. A. (1999). Independent development of sensory and motor
innervation patterns in embryonic chick hindlimbs. Dev. Biol. 208, 324-336.
Wang, L., Klein, R., Zheng, B. and Marquardt, T. (2011). Anatomical coupling of
sensory and motor nerve trajectory via axon tracking. Neuron 71, 263-277.
Wenner, P. and Frank, E. (1995). Peripheral target specification of synaptic
connectivity of muscle spindle sensory neurons with spinal motoneurons.
J. Neurosci. 15, 8191-8198.
Yang, X., Arber, S., William, C., Li, L., Tanabe, Y., Jessell, T. M., Birchmeier, C.
and Burden, S. J. (2001). Patterning of muscle acetylcholine receptor gene
expression in the absence of motor innervation. Neuron 30, 399-410.
Yip, J. W. (1990). Identification of location and timing of guidance cues in
sympathetic preganglionic axons of the chick. J. Neurosci. 10, 2476-2484.
Zhang, J., Lefebvre, J. L., Zhao, S. and Granato, M. (2004). Zebrafish unplugged
reveals a role for muscle-specific kinase homologs in axonal pathway choice.
Nature. Neurosci. 7, 1303-1309.
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E
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